Thrombin is a serine protease which serves as an active component in several hemostasis products. For example, fibrin sealants typically comprise a fibrinogen component and a thrombin component. When both components are mixed (e.g. when applied to a bleeding wound or surgical incision) thrombin cleaves fibrinogen and a fibrin polymer is formed. Concentrated purified thrombin in liquid form displays a reduction in activity during prolonged storage, primarily as a result of autolysis.
Hemostatic formulations containing liquid thrombin have special handling requirements in order to maintain thrombin's biologic activity and prevent autolytic degradation. For example, liquid thrombin requires freezing or the addition of protease inhibitors to maintain shelf-life stability. The shortcoming of the liquid thrombin solutions in use today are manifold: In the clinic and operating room, freezing is expensive and not always feasible, and promiscuous protease inhibitors may adversely affect activity of thrombin and other proteases in the hemostasis pathway once the fibrin sealant or the thrombin is applied.
Liquid thrombin preparations may be made into a lyophilized medical preparation, which is used after dissolving at the time of use. However, liquid preparations are advantageous compared with the lyophilized preparations in that they can be easily administered without the additional step of dissolving in a solvent prior to use. Also, the lyophilization step is costly and time consuming and may result in loss of yield.
Known compositions and methods for stabilizing thrombin are unsatisfactory and include the following: inclusion of various non-specific components (e.g. bulk carrier proteins such as albumins, different stabilizing sugars, general protease inhibitors etc.); formulation of the thrombin with inhibitors of thrombin activity, which although may be efficient, also inactivate or inhibit the thrombin, thereby reducing its effectiveness; and formulation of a low thrombin concentration solution, thereby necessitating administration of larger amounts of the formulation.
Several publications relate to stabilization of thrombin: for example International Patent Application Publication No. WO2008157304; U.S. Pat. Nos. 4,409,334; 7,351,561 and 8,394,372; U.S. Patent Application No. 20080311104; and European Patent Nos. EP0277096 B1 and EP 0478827 B1.
There remains a need for thrombin specific compounds useful to stabilize thrombin from autolytic degradation. Preferably, the compounds are reversible thrombin inhibitors which may be used with a concentrated liquid thrombin formulation.